The invention relates nucleoside prodrugs which are inhibitors of HCV replication. In particular, the invention is concerned with the use of acylated pyrimidine nucleoside compounds which provide improved drug absorption when the nucleoside is administered orally.
Hepatitis C virus is a major health problem and the leading cause of chronic liver disease throughout the world. (Boyer, N. et al. J. Hepatol. 2000 32:98-112). Patients infected with HCV are at risk of developing cirrhosis of the liver and subsequent hepatocellular carcinoma and, hence, HCV is the major indication for liver transplantation.
According to the World Health Organization, there are more than 200 million infected individuals worldwide, with at least 3 to 4 million people being infected each year. Once infected, about 20% of people clear the virus, but the rest can harbor HCV the rest of their lives. Ten to twenty percent of chronically infected individuals eventually develop liver-destroying cirrhosis or cancer. The viral disease is transmitted parenterally by contaminated blood and blood products, contaminated needles, or sexually and vertically from infected mothers or carrier mothers to their offspring. Current treatments for HCV infection, which are restricted to immunotherapy with recombinant interferon-α alone or in combination with the nucleoside analog ribavirin, are of limited clinical benefit as resistance develops rapidly. There is an urgent need for improved therapeutic agents that effectively combat chronic HCV infection
HCV has been classified as a member of the virus family Flaviviridae that includes the genera flaviviruses, pestiviruses, and hepaciviruses which includes hepatitis C viruses (Rice, C. M., Flaviviridae: The viruses and their replication, in: Fields Virology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 30, 931-959, 1996). HCV is an enveloped virus containing a positive-sense single-stranded RNA genome of approximately 9.4 kb. The viral genome consists of a 5′-untranslated region (UTR), a long open reading frame (ORF) encoding a polyprotein precursor of-approximately 3011 amino acids, and a short 3′ UTR. The 5′ UTR is the most highly conserved part of the HCV genome and is important for the initiation and control of polyprotein translation.
Genetic analysis of HCV has identified six main genotypes showing a >30% divergence in their DNA sequence. Each genotype contains a series of more closely related subtypes which show a 20-25% divergence in nucleotide sequences (Simmonds, P. 2004 J. Gen. Virol. 85:3173-88). More than 30 subtypes have been distinguished. In the US approximately 70% of infected individuals have Type 1a and 1b infection. Type 1b is the most prevalent subtype in Asia. (X. Forns and J. Bukh, Clinics in Liver Disease 1999 3:693-716; J. Bukh et al., Semin. Liv. Dis. 1995 15:41-63). Unfortunately Type 1 infections are more resistant to therapy than either the type 2 or 3 genotypes (N. N. Zein, Clin. Microbiol. Rev., 2000 13:223-235).
The genetic organization and polyprotein processing of the nonstructural protein portion of the ORF of pestiviruses and hepaciviruses is very similar. These positive stranded RNA viruses possess a single large ORF encoding all the viral proteins necessary for virus replication. These proteins are expressed as a polyprotein that is co- and post-translationally processed by both cellular and virus-encoded proteinases to yield the mature viral proteins. The viral proteins responsible for the replication of the viral genome RNA are located within approximately the carboxy-terminal. Two-thirds of the ORF are termed nonstructural (NS) proteins. For both the pestiviruses and hepaciviruses, the mature nonstructural (NS) proteins, in sequential order from the amino-terminus of the nonstructural protein coding region to the carboxy-terminus of the ORF, consist of p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B.
The NS proteins of pestiviruses and hepaciviruses share sequence domains that are characteristic of specific protein functions. For example, the NS3 proteins of viruses in both groups possess amino acid sequence motifs characteristic of serine proteinases and of helicases (Gorbalenya et al. Nature 1988 333:22; Bazan and Fletterick Virology 1989 171:637-639; Gorbalenya et al. Nucleic Acid Res. 1989 17.3889-3897). Similarly, the NS5B proteins of pestiviruses and hepaciviruses have the motifs characteristic of RNA-directed RNA polymerases (Koonin, E. V. and Doija, V. V. Crit. Rev. Biochem. Molec. Biol. 1993 28:375-430).
The actual roles and functions of the NS proteins of pestiviruses and hepaciviruses in the lifecycle of the viruses are directly analogous. In both cases, the NS3 serine proteinase is responsible for all proteolytic processing of polyprotein precursors downstream of its position in the ORF (Wiskerchen and Collett Virology 1991 184:341-350; Bartenschlager et al. J. Virol. 1993 67:3835-3844; Eckart et al. Biochem. Biophys. Res. Comm. 1993 192:399-406; Grakoui et al. J. Virol. 1993 67:2832-2843; Grakoui et al. Proc. Natl. Acad. Sci. USA 1993 90:10583-10587; Ilijikata et al. J. Virol. 1993 67:4665-4675; Tome et al. J. Virol. 1993 67:4017-4026). The NS4A protein, in both cases, acts as a cofactor with the NS3 serine protease (Bartenschlager et al. J. Virol. 1994 68:5045-5055; Failla et al. J. Virol. 1994 68: 3753-3760; Xu et al. J Virol. 1997 71:53 12-5322). The NS3 protein of both viruses also functions as a helicase (Kim et al. Biochem. Biophys. Res. Comm. 1995 215: 160-166; Jin and Peterson Arch. Biochem. Biophys. 1995, 323:47-53; Warrener and Collett J. Virol. 1995 69:1720-1726). Finally, the NS5B proteins of pestiviruses and hepaciviruses have the predicted RNA-directed RNA polymerases activity (Behrens et al. EMBO 1996 15:12-22; Lechmann et al. J. Virol. 1997 71:8416-8428; Yuan et al. Biochem. Biophys. Res. Comm. 1997 232:231-235; Hagedorn, PCT WO 97/12033; Zhong et al. J. Virol. 1998 72:9365-9369).
Currently there are a limited number of approved therapies are currently available for the treatment of HCV infection. New and existing therapeutic approaches to treating HCV and inhibition of HCV NS5B polymerase have been reviewed: R. G. Gish, Sem. Liver. Dis., 1999 19:5; Di Besceglie, A. M. and Bacon, B. R., Scientific American, October: 1999 80-85; G. Lake-Bakaar, Current and Future Therapy for Chronic Hepatitis C Virus Liver Disease, Curr. Drug Targ. Infect Dis. 2003 3(3):247-253; P. Hoffmann et al., Recent patents on experimental therapy for hepatitis C virus infection (1999-2002), Exp. Opin. Ther. Patents 2003 13(11):1707-1723; F. F. Poordad et al. Developments in Hepatitis C therapy during 2000-2002, Exp. Opin. Emerging Drugs 2003 8(1):9-25; M. P. Walker et al., Promising Candidates for the treatment of chronic hepatitis C, Exp. Opin. Investig. Drugs 2003 12(8): 1269-1280; S.-L. Tan et al., Hepatitis C Therapeutics: Current Status and Emerging Strategies, Nature Rev. Drug Discov. 2002 1:867-881; R. De Francesco et al. Approaching a new era for hepatitis C virus therapy: inhibitors of the NS3-4A serine protease and the NS5B RNA-dependent RNA polymerase, Antiviral Res. 2003 58:1-16; Q. M. Wang et al. Hepatitis C virus encoded proteins: targets for antiviral therapy, Drugs of the Future 2000 25(9):933-8-944; J. A. Wu and Z. Hong, Targeting NS5B-Dependent RNA Polymerase for Anti-HCV Chemotherapy Cur. Drug Targ.-Inf. Dis. 2003 3:207-219. The reviews cite compounds presently in various stages of the development process. Combination therapy with two or three agents directed to the same or different targets has become standard therapy to avoid or slow the development of resistant strains of a virus and the compounds disclosed in the above reviews could be used in combination therapy with compounds of the present invention and these reviews are hereby incorporated by reference in their entirety.
                1a: R═C(═O)NH2         1b: R═C(═NH+)NH2         
Ribavirin (1a; 1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-1H-[1,2,4]triazole-3-carboxylic acid amide; VIRAZOLE®) is a synthetic, non-interferon-inducing, broad spectrum antiviral nucleoside analog. Ribavirin has in vitro activity against several DNA and RNA viruses including Flaviviridae (Gary L. Davis, Gastroenterology 2000 118:S104-S114). In monotherapy ribavirin reduces serum amino transferase levels to normal in 40% of patients, but it does not lower serum levels of HCV-RNA. Ribavirin also exhibits significant toxicity and is known to induce anemia. Ribavirin is an inhibitor of inosine monophosphate dehydrogenase. Ribovirin is not approved in monotherapy against HCV but the compound is approved in combination therapy with interferon α-2a and interferon α-2b. Viramidine 1b is a prodrug converted to 1a in hepatocytes.
Interferons (IFNs) have been available for the treatment of chronic hepatitis for nearly a decade. IFNs are glycoproteins produced by immune cells in response to viral infection. Two distinct types of interferon are recognized: Type 1 includes several interferon alphas and one interferon β, type 2 includes interferon γ. Type 1 interferon is produced mainly by infected cells and protects neighboring cells from de novo infection. IFNs inhibit viral replication of many viruses, including HCV, and when used as the sole treatment for hepatitis C infection, IFN suppresses serum HCV-RNA to undetectable levels. Additionally, IFN normalizes serum amino transferase levels. Unfortunately, the effects of IFN are temporary. Cessation of therapy results in a 70% relapse rate and only 10-15% exhibit a sustained virological response with normal serum alanine transferase levels. (L.-B. Davis, supra)
One limitation of early IFN therapy was rapid clearance of the protein from the blood. Chemical derivatization of IFN with polyethyleneglycol (PEG) has resulted in proteins with substantially improved pharmacokinetic properties. PEGASYS® is a conjugate interferon α-2a and a 40 kD branched mono-methoxy PEG and PEG-INTRON® is a conjugate of interferon α-2b and a 12 kD mono-methoxy PEG. (B. A. Luxon et al., Clin. Therap. 2002 24(9):13631383; A. Kozlowski and J. M. Harris, J. Control. Release, 2001 72:217-224).
Interferon α-2a and interferon α-2b are currently approved as monotherapy for the treatment of HCV. ROFERON-A® (Roche) is the recombinant form of interferon α-2a. PEGASYS® (Roche) is the pegylated (i.e. polyethylene glycol modified) form of interferon α-2a. INTRON-A® (Schering Corporation) is the recombinant form of Interferon α-2b, and PEG-INTRON® (Schering Corporation) is the pegylated form of interferon α-2b.
Other forms of interferon α, as well as interferon β, γ, τ and ω are currently in clinical development for the treatment of HCV. For example, INFERGEN® (interferon alphacon-1) by InterMune, OMNIFERON® (natural interferon) by Viragen, ALBUFERON® by Human Genome Sciences, REBIF® (interferon β-1a) by Ares-Serono, Omega Interferon by BioMedicine, Oral Interferon Alpha by Amarillo Biosciences, and interferon γ, interferon τ, and interferon γ-1b by InterMune are in development.
Combination therapy of HCV with ribavirin and interferon-α currently represent the optimal therapy. Combining ribavirin and PEG-IFN (infra) results in a sustained viral response in 54-56% of patients. The SVR approaches 80% for type 2 and 3 HCV. (Walker, supra) Unfortunately, the combination also produces side effects which pose clinical challenges. Depression, flu-like symptoms and skin reactions are associated with subcutaneous IFN-α and hemolytic anemia is associated with sustained treatment with ribavirin.
Other macromolecular compounds currently in preclinical or clinical development for treatment of hepatitis C virus infection include: Interleukin-10 by Schering-Plough, IP-SO1 by Intemeuron, Merimebodib (VX-497) by Vertex, HEPTAZYME® by RPI, IDN-6556 by Idun Pharma., XTL-002 by XTL., HCV/MFS9 by Chiron, CIVACIR® (hepatitis C Immune Globulin) by NABI, ZADAXIN® (thymosin α-l) by SciClone, thymosin plus pegylated interferon by SciClone, CEPLENE®; a therapeutic vaccine directed to E2 by Innogenetics, therapeutic vaccine by Intercell, therapeutic vaccine by Epimmune/Genencor, a therapeutic vaccine by Merix, a therapeutic vaccine, Chron-VacC, by Tripep.
Other macromolecular approaches include ribozymes targeted at HCV RNA. Ribozymes are short naturally occurring molecules with endonuclease activity that catalyze the sequence-specific cleavage of RNA. An alternate approach is the use of antisense oligonucleotides bind to RNA and stimulate RNaseH mediated cleavage.
A number of potential molecular targets for drug development as anti-HCV therapeutics have now been identified including, but not limited to, the NS2-NS3 autoprotease, the N3 protease, the N3 helicase and the NS5B polymerase. The RNA-dependent RNA polymerase is absolutely essential for replication of the single-stranded, positive sense, RNA genome and this enzyme has elicited significant interest among medicinal chemists.
Nucleoside inhibitors of NS5B polymerase can act either as a non-natural substrate that results in chain termination or as a competitive inhibitor which competes with nucleotide binding to the polymerase. To function as a chain terminator the nucleoside analog must be taken up be the cell and converted in vivo to a triphosphate to compete for the polymerase nucleotide binding site. This conversion to the triphosphate is commonly mediated by cellular kinases which imparts additional structural requirements on a potential nucleoside polymerase inhibitor. Unfortunately, this limits the direct evaluation of nucleosides as inhibitors of HCV replication to cell-based assays capable of in situ phosphorylation.
    B=adenine, thymidine, uracil, cytidine, guanine and hypoxanthine
In WO 01 90121 published Nov. 29, 2001, J.-P. Sommadossi and P. Lacolla disclose and exemplify the anti-HCV polymerase activity of 1′-alkyl- and 2′-alkyl nucleosides of formulae 2 and 3. In WO 01/92282, published Dec. 6, 2001, J.-P. Sommadossi and P. Lacolla disclose and exemplify treating Flaviviruses and Pestiviruses with 1′-alkyl- and 2′-alkyl nucleosides of formulae 2 and 3. In WO 03/026675 published Apr. 3, 2003, G. Gosselin discloses 4′-alkyl nucleosides 4 for treating Flaviviruses and Pestiviruses.
In WO2004003000 published Jan. 8, 2004, J.-P. Sommadossi et al. disclose 2′- and 3′ prodrugs of 1′-, 2′-, 3′- and 4′-substituted β-D and β-L nucleosides. In WO 2004/002422 published Jan. 8, 2004, 2′-C-methyl-3′-O-valine ester ribofuransyl cytidine for the treatment of Flaviviridae infections. Idenix has reported clinical trials for a related compound NM283 which is believed to be the valine ester 5 of the cytidine analog 2 (B=cytosine). In WO 2004/002999 published Jan. 8, 2004, J.-P. Sommadossi et al. disclose a series of 2′ or 3′prodrugs of 1′, 2′, 3′, or 4′ branched nucleosides for the treatment of flavivirus infections including HCV infections.
In WO2004/046331 published Jun. 3, 2004, J.-P. Sommadossi et al. disclose 2′-branched nucleosides and Flaviviridae mutation. In WO03/026589 published Apr. 3, 2003 G. Gosselin et al. disclose methods of treating hepatitis C virus using 4′-modified nucleosides. In WO2005009418 published Feb. 3, 2005, R. Storer et al. disclose purine nucleoside analogues for treatment of diseases caused by Flaviviridae including HCV.
Other patent applications disclose the use of certain nucleoside analogs to treat hepatitis C virus infection. In WO 01/32153 published May 10, 2001, R. Storer discloses nucleosides derivatives for treating viral diseases. In WO 01/60315 published Aug. 23, 2001, H. Ismaili et al., disclose methods of treatment or prevention of Flavivirus infections with nucleoside compounds. In WO 02/18404 published Mar. 7, 2002, R. Devos et al. disclose 4′-substituted nucleotides for treating HCV virus. In WO 01/79246 published Oct. 25, 2001, K. A. Watanabe disclose 2′- or 3′-hydroxymethyl nucleoside compounds for the treatment of viral diseases. In WO 02/32920 published Apr. 25, 2002 and in WO 02/48 165 published Jun. 20, 2002 L. Stuyver et al. disclose nucleoside compounds for the treatment of viral diseases.

In WO 03/105770 published Dec. 24, 2003, B. Bhat et al. disclose a series of carbocyclic nucleoside derivatives that are useful for the treatment of HCV infections. In WO 2004/007512 published Jan. 22, 2003 B. Bhat et al. disclose nucleoside compounds that inhibit of RNA-dependent RNA viral polymerase. The nucleosides disclosed in this publication are primarily 2′-methyl-2′-hydroxy substituted nucleosides. In WO 2002/057425 published Jul. 25, 2002 S. S. Carroll et al. disclose nucleoside derivatives which inhibitor of RNA-dependent viral polymerase and methods of treating HCV infection. In WO02/057287 published Jul. 25, 2002, S. S. Carroll et al. disclose related 2α-methyl and 2β-methylribose derivatives wherein the base is an optionally substituted 7H-pyrrolo[2,3-d]pyrimidine radical 6. The same application discloses one example of a 3β-methyl nucleoside. S. S. Carroll et al. (J. Biol. Chem. 2003 278(14):11979-11984) disclose inhibition of HCV polymerase by 2′-O-methylcytidine (6a). In WO 2004/009020 published Jan. 29, 2004, D. B. Olsen et al. disclose a series of thionucleoside derivatives as inhibitors of RNA dependent RNA viral polymerase.
PCT Publication No. WO 99/43691 to Emory University, entitled “2′-Fluoronucleosides” discloses the use of certain 2′-fluoronucleosides to treat HCV. U.S. Pat. No. 6,348,587 to Emory University entitled “2′-fluoronucleosides” discloses a family of 2′-fluoronucleosides useful for the treatment of hepatitis B, HCV, HIV and abnormal cellular proliferation. Both configurations of the 2′ fluoro substituent are disclosed.
Eldrup et al. (Oral Session V, Hepatitis C Virus, Flaviviridae; 16th International Conference on Antiviral Research (Apr. 27, 2003, Savannah, Ga.)) described the structure activity relationship of 2′-modified nucleosides for inhibition of HCV.
Bhat et al. (Oral Session V, Hepatitis C Virus, Flaviviridae; 16th International Conference on Antiviral Research (Apr. 27, 2003, Savannah, Ga.); p A75) describe the synthesis and pharmacokinetic properties of nucleoside analogues as possible inhibitors of HCV RNA replication. The authors report that 2′-modified nucleosides demonstrate potent inhibitory activity in cell-based replicon assays.
Olsen et al. (Oral Session V, Hepatitis C Virus, Flaviviridae; 16th International Conference on Antiviral Research (Apr. 27, 2003, Savannah, Ga.) p A76) also described the effects of the 2′-modified nucleosides on HCV RNA replication.
Non-nucleoside allosteric inhibitors of HIV reverse transcriptase have proven effective therapeutics alone and in combination with nucleoside inhibitors and with protease inhibitors. Several classes of non-nucleoside HCV NS5B inhibitors have been described and are currently at various stages of development including: benzimidazoles, (H. Hashimoto et al. WO 01/47833, H. Hashimoto et al. WO 03/000254, P. L. Beaulieu et al. WO 03/020240 A2; P. L. Beaulieu et al. U.S. Pat. No. 6,448,281 B1; P. L. Beaulieu et al. WO 03/007945 A1); indoles, (P. L. Beaulieu et al. WO 03/0010141 A2); benzothiadiazines, e.g., 7, (D. Dhanak et al. WO 01/85172 A1; D. Dhanak et al. WO 03/037262 A2; K. J. Duffy et al. WO03/099801 A1, D. Chai et al. WO 2004052312, D. Chai et al. WO2004052313, D. Chai et al. WO02/098424, J. K. Pratt et al. WO 2004/041818 A1; J. K. Pratt et al. WO 2004/087577 A1), thiophenes, e.g., 8, (C. K. Chan et al. WO 02/100851);
benzothiophenes (D. C. Young and T. R. Bailey WO 00/18231); β-ketopyruvates (S. Attamura et al. U.S. Pat. No. 6,492,423 B1, A. Attamura et al. WO 00/06529); pyrimidines (C. Gardelli et al. WO 02/06246 A1); pyrimidinediones (T. R. Bailey and D. C. Young WO 00/13708); triazines (K.-H. Chung et al. WO 02/079187 A1); rhodanine derivatives (T. R. Bailey and D. C. Young WO 00/10573, J. C. Jean et al. WO 01/77091 A2); 2,4-dioxopyrans (R. A. Love et al. EP 256628 A2); phenylalanine derivatives (M. Wang et al. J. Biol. Chem. 2003 278:2489-2495).
The NS3 protease has emerged as a major target for discovery of new anti-HCV therapy. In WO 98/22496 published May 28, 1998, M. R. Attwood et al. have disclosed mechanism-base active site inhibitors of the protease (M. R. Attwood et al, Antiviral Chemistry and Chemotherapy 1999 10:259-273; M. R. Attwood et al., Preparation and use of amino acid derivatives as anti-viral agents, German Patent Pub. DE 19914474). In WO98/17679 published Apr. 30, 1998 R. D. Tung et al. disclosed mechanism-based peptide inhibitors on the NS3 protease.
In WO99/07734 published Feb. 18, 1999 and in WO00/09543 published Aug. 9, 1999, M Llinas-Brunet et al. disclose peptide inhibitors of the protease. In WO00/59929 published Oct. 12, 2000, Y. S. Tsantrizos et al. disclose macrocyclic tripeptides which are potent inhibitors of the HCV NS3 protease. A series of related patents from Boehringer-Ingleheim disclose related protease inhibitors and have lead to the identification of tripeptide derivative BILN 2061 (M. Llinas-Brunet et al. Biorg. Med. Chem. Lett. 2000 10(20):2267-70; J. Med Chem. 2004 47(26):6584-94; J. Med. Chem. 2004 47(7):1605-1608; Angew. Chem. Int. Ed. Eng. 2003 42(12):1356-60).
Other tripeptide inhibitors identified by Bristol-Myers Squibb have been disclosed, inter alia, in WO03/099274 published Dec. 4, 2003, in WO2004/032827 published Apr. 22, 2004, in WO03/053349 published Jul. 3, 2003, in WO2005/046712 published May 26, 2005 and in WO2005/051410 published Jun. 9, 2005. In WO2004/072234 published Aug. 26, 2004 and in WO2004/093798 published Nov. 4, 2004 further tripeptide protease inhibitors were disclosed by Enanta Pharmaceuticals. In WO2005/037214 published Apr. 28, 2005 L. M. Blatt et al. disclose still other tripeptide derivatives that inhibit HCV NS3 protease. In WO2005/030796 published Apr. 7, 2005, S. Venkatraman et al. disclose macrocyclic inhibitors of HCV NS3 serine protease. In WO 2005/058821 published Jun. 30, 2005, F. Velazquez et al. disclose inhibitors of HCV NS3/NS4a serine protease. In WO02/48172 published Jun. 20, 2002, Z. Zhu disclose diaryl peptides as inhibitors of NS3 protease. In WO02/08187 and in WO02/08256 both published Jan. 31, 2002, A. Saksena et al. disclose peptide inhibitors of HCV NS3 protease. In WO02/08251 published Jan. 31, 2002 M. Lim-Wilby et al. disclose peptide inhibitors of the NS3 protease. In U.S. Pat. No. 6,004,933 published Dec. 21, 1999, L. W. Spruce et al. disclose heterocyclic peptide derivatives which inhibit cysteine proteases including HCV endopeptidase.
Non-substrate-based NS3 protease inhibitors such as 2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al., BBRC 1997 238:643-647; Sudo K. et al. Antiviral Chemistry and Chemotherapy 1998 9:186), including RD3-4082 and RD3-4078, the former substituted on the amide with a 14 carbon chain and the latter processing a para-phenoxyphenyl group are also being investigated.
SCH 68631, a phenanthrenequinone, is an HCV protease inhibitor (Chu M. et al. Tetrahedron Lett. 1996 37:7229-7232). In another example by the same authors, SCH 351633, isolated from the fungus Penicillium griseofulvum, was identified as a protease inhibitor (Chu M. et al., Bioorg. Med. Chem. Lett. 1999 9:1949-1952). Nanomolar potency against the HCV NS3 protease enzyme has been achieved by the design of selective inhibitors based on the macromolecule eglin c. Eglin c, isolated from leech, is a potent inhibitor of several serine proteases such as S. griseus proteases A and B, α-chymotrypsin, chymase and subtilisin (Qasim M. A. et al., Biochemistry 1997 36:1598-1607).
Thiazolidine derivatives which show relevant inhibition in a reverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5B substrate (Sudo K. et al. Antiviral Research 1996 32:9-18), especially compound RD-1-6250, possessing a fused cinnamoyl moiety substituted with a long alkyl chain, RD4 6205 and RD4 6193. Thiazolidines and benzanilides were identified by N. Kakiuchi et al. in FEBS Let. 1998 421:217-220 and N. Takeshita et al. Anal. Biochem. 1997 247:242-246.
Imidazolidinones as NS3 serine protease inhibitors of HCV are disclosed in WO 02/08198 to Schering Corporation published Jan. 31, 2002 and in WO 02/48157 to Bristol Myers Squibb published Jun. 20, 2002. In WO02/48116 published Jun. 20, 2002 P. Glunz et al. disclose pyrimidinone inhibitors of NS3 protease.
Other enzymatic targets for anti-HCV include the HCV IRES site (Internal Ribosomal Entry Site) and HCV helicase. IRES inhibitors have been reported by Immusol, Rigel Pharmaceuticals (R803) and by Anadys (ANA 245 and ANA 246). Vertex has disclosed an HCV helicase inhibitor.
Combination therapy which can suppress resistant mutant strains, has become well established approach to anti-viral chemotherapy. The nucleoside inhibitors disclosed herein can be combined with other nucleoside HCV polymerase inhibitors, non-nucleoside HCV polymerase inhibitors, and HCV protease inhibitors. As other classes of HCV drugs, e.g. viral entry inhibitors, helicase inhibitors, IRES inhibitors, ribozymes and antisense oligonucleotides emerge and are developed they will also be excellent candidates for combination therapy. Interferon derivatives have already been successfully combined with ribavirin and interferons and chemically modified interferons will be useful in combination with the nucleosides herein disclosed.